Structural determinants of the direct inhibition of GIRK channels by Sigma-1 receptor antagonist.

G-protein-gated inward rectifier K+ (GIRK) channels play a critical role in the regulation of the excitability of cardiomyocytes and neurons and include GIRK1, GIRK2, GIRK3 and GIRK4 subfamily members. BD1047 dihydrobromide (BD1047) is one of the representative antagonists of the multifunctional Sigma-1 receptor (S1R). In the analysis of the effect of BD1047 on the regulation of Gi-coupled receptors by S1R using GIRK channel as an effector, we observed that BD1047, as well as BD1063, directly inhibited GIRK currents even in the absence of S1R and in a voltage-independent manner. Thus, we aimed to clarify the effect of BD1047 on GIRK channels and identify the structural determinants. By electrophysiological recordings in Xenopus oocytes, we observed that BD1047 directly inhibited GIRK channel currents, producing a much stronger inhibition of GIRK4 compared to GIRK2. It also inhibited ACh-induced native GIRK current in isolated rat atrial myocytes. Chimeric and mutagenesis studies of GIRK2 and GIRK4 combined with molecular docking analysis demonstrated the importance of Leu77 and Leu84 within the cytoplasmic, proximal N-terminal region and Glu147 within the pore-forming region of GIRK4 for inhibition by BD1047. The activator of GIRK channels, ivermectin, competed with BD1047 at Leu77 on GIRK4. This study provides us with a novel inhibitor of GIRK channels and information for developing pharmacological treatments for GIRK4-associated diseases.

Structural insights in the permeation mechanism of an activated GIRK2 channel.

G protein-gated inwardly rectifying potassium (GIRK) channels play a significant role in physiopathology by the regulation of cell excitability. This regulation depends on the K+ ion conduction induced by structural constrictions: the selectivity filters (SFs), helix bundle crossings (HBCs), and G-loop gates. To explore why no permeation occurred when the constrictions were kept in the open state, a 4-K+-related occupancy mechanism was proposed. Unfortunately, this hypothesis was neither assessed, nor was the energetic characteristics presented. To identify the permeation mechanism on an atomic level, all-atom molecular dynamic (MD) simulations and a coupled quantum mechanics and molecular mechanics (QM/MM) method were used for the GIRK2 mutant R201A. It was found that the R201A had a moderate conductive capability in the presence of PIP2. Furthermore, the 4-K+ group of ions was found to dominate the conduction through the activated HBC gate. This shielding-like mechanism was assessed by the potential energy barrier along the conduction pathway. Mutation studies did further support the assumption that E152 was responsible for the mechanism. Moreover, E152 was most probably facilitating the inflow of ions from the SF to the cavity. On the contrary, N184 had no remarkable effect on this mechanism, except for the conduction efficiency. These findings highlighted the necessity of a multi-ion distribution for the conduction to take place, and indicated that the K+ migration was not only determined by the channel conductive state in the GIRK channel. The here presented multi-ion permeation mechanism may help to provide an effective way to regulate the channelopathies.

Mechanism of PKCε regulation of cardiac GIRK channel gating.

G-protein-gated inwardly rectifying potassium (GIRK) channel activity is regulated by the membrane phospholipid, phosphatidylinositol-4,5-bisphosphate (PI 4,5P2). Constitutive activity of cardiac GIRK channels in atrial myocytes, that is implicated in atrial fibrillation (AF), is mediated via a protein kinase C-ε (PKCε)-dependent mechanism. The novel PKC isoform, PKCε, is reported to enhance the activity of cardiac GIRK channels. Here, we report that PKCε stimulation leads to activation of GIRK channels in mouse atria and in human stem cell-derived atrial cardiomyocytes (iPSCs). We identified residue GIRK4(S418) which when mutated to Ala abolished, or to Glu, mimicked the effects of PKCε on GIRK currents. PKCε strengthened the interactions of the cardiac GIRK isoforms, GIRK4 and GIRK1/4 with PIP2, an effect that was reversed in the GIRK4(S418A) mutant. This mechanistic insight into the PKCε-mediated increase in channel activity because of GIRK4(S418) phosphorylation, provides a precise druggable target to reverse AF-related pathologies due to GIRK overactivity.

A selectivity filter mutation provides insights into gating regulation of a K+ channel.

G-protein coupled inwardly rectifying potassium (GIRK) channels are key players in inhibitory neurotransmission in heart and brain. We conducted molecular dynamics simulations to investigate the effect of a selectivity filter (SF) mutation, G154S, on GIRK2 structure and function. We observe mutation-induced loss of selectivity, changes in ion occupancy and altered filter geometry. Unexpectedly, we reveal aberrant SF dynamics in the mutant to be correlated with motions in the binding site of the channel activator Gßγ. This coupling is corroborated by electrophysiological experiments, revealing that GIRK2wt activation by Gßγ reduces the affinity of Ba2+ block. We further present a functional characterization of the human GIRK2G154S mutant validating our computational findings. This study identifies an allosteric connection between the SF and a crucial activator binding site. This allosteric gating mechanism may also apply to other potassium channels that are modulated by accessory proteins.

Entropy in the Molecular Recognition of Membrane Protein-Lipid Interactions.

Understanding the molecular driving forces that underlie membrane protein-lipid interactions requires the characterization of their binding thermodynamics. Here, we employ variable-temperature native mass spectrometry to determine the thermodynamics of lipid binding events to the human G-protein-gated inward rectifier potassium channel, Kir3.2. The channel displays distinct thermodynamic strategies to engage phosphatidylinositol (PI) and phosphorylated forms thereof. The addition of a 4'-phosphate to PI results in an increase in favorable entropy. PI with two or more phosphates exhibits more complex binding, where lipids appear to bind two nonidentical sites on Kir3.2. Remarkably, the interaction of 4,5-bisphosphate PI with Kir3.2 is solely driven by a large, favorable change in entropy. Installment of a 3'-phosphate to PI(4,5)P2 results in an altered thermodynamic strategy. The acyl chain of the lipid has a marked impact on binding thermodynamics and, in some cases, enthalpy becomes favorable.

Insight into the Phospholipid-Binding Preferences of Kir3.4.

The G-protein-gated inwardly rectifying potassium channel 4 (Kir3.4) subunit forms functional tetramers. Previous studies have established that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is required for Kir3.4 function. However, the binding preferences of Kir3.4 for the headgroup and acyl chains of phosphorylated phosphatidylinositides (PIPs) and other lipids are not well understood. Here, the interactions between full-length, human Kir3.4 and lipids are characterized using native mass spectrometry (MS) in conjunction with a soluble fluorescent lipid-binding assay. Kir3.4 displays binding preferences for PIPs, and, in some cases, the degree of binding is influenced by the type of acyl chains. The interactions between Kir3.4 and PIPs are weaker in comparison to full-length, human Kir3.2. The binding of PI(4,5)P2 modified with a fluorophore to Kir3.2 can be enhanced by other lipids, such as phosphatidylcholine. Introduction of S143T, a mutation that enhances Kir3.4 activity, results in an overall reduction in the channel binding PIPs. In contrast, the D223N mutant of Kir3.4 that mimics the sodium-bound state exhibited stronger binding for PI(4,5)P2, particularly for those with 18:0-20:4 acyl chains. Taken together, these results provide additional insight into the interaction between Kir3.4 and lipids that are important for channel function.

Characterization of a mutated KCNJ5 gene, G387R, in unilateral primary aldosteronism.

Somatic mutation in the KCNJ5 gene is a common driver of autonomous aldosterone overproduction in aldosterone-producing adenomas (APA). KCNJ5 mutations contribute to a loss of potassium selectivity, and an inward Na+ current could be detected in cells transfected with mutated KCNJ5. Among 223 unilateral primary aldosteronism (uPA) individuals with a KCNJ5 mutation, we identified 6 adenomas with a KCNJ5 p.Gly387Arg (G387R) mutation, previously unreported in uPA patients. The six uPA patients harboring mutant KCNJ5-G387R were older, had a longer hypertensive history, and had milder elevated preoperative plasma aldosterone levels than those APA patients with more frequently detected KCNJ5 mutations. CYP11B2 immunohistochemical staining was only positive in three adenomas, while the other three had co-existing multiple aldosterone-producing micronodules. The bioinformatics analysis predicted that function of the KCNJ5-G387R mutant channel could be pathological. However, the electrophysiological experiment demonstrated that transfected G387R mutant cells did not have an aberrantly stimulated ion current, with lower CYP11B2 synthesis and aldosterone production, when compared to that of the more frequently detected mutant KCNJ5-L168R transfected cells. In conclusion, mutant KCNJ5-G387R is not a functional KCNJ5 mutation in unilateral PA. Compared with other KCNJ5 mutations, the observed mildly elevated aldosterone expression actually hindered the clinical identification of clinical unilateral PA. The KCNJ5-G387R mutation needs to be distinguished from functional KCNJ5 mutations during genomic analysis in APA evaluation because of its functional silence.

A double bilayer to study the nonequilibrium environmental response of GIRK2 in complex states.

G protein-gated inwardly rectifying potassium (GIRK) channels play essential roles in electrical signaling in neurons and muscle cells. Nonequilibrium environments provide crucial driving forces behind many cellular events. Here, we apply the antiparallel alignment double bilayer model to study GIRK2 in response to the time-dependent membrane potential. Using molecular dynamics and umbrella sampling, we examined the time-dependent environmental impact on the ion conduction, energy basis, and primary motions of GIRK2 in different complex states with phosphatidylinositol-4,5-bisphosphate (PIP2) and G-protein ßγ subunits (Gßγ). The antiparallel alignment double bilayer model enables us to study the transport performance in inward and outward K+ and mixed K+ and Na+. We obtained the recoverable discharge process of GIRK2 complexed with both PIP2 and Gßγ, compared with occasional conduction under PIP2-only regulation. Calculations of potential of mean force suggest different regulation by the helix bundle crossing (HBC) gate and G-loop gate regarding different complex states and under a membrane potential. In a nonequilibrium environment, distinct functional rocking motions of GIRK2 were identified under strengthened correlations between the transmembrane helices and downstream cytoplasmic domains with binding of PIP2, cations, and Gßγ. The findings suggest the potential domain motions and dynamics associated with a nonequilibrium environment and highlight the application of the antiparallel alignment double bilayer model to investigate factors in an asymmetric environment.

Multifaceted Regulation of Potassium-Ion Channels by Graphene Quantum Dots.

Graphene quantum dots (GQDs) are emerging as a versatile nanomaterial with numerous proposed biomedical applications. Despite the explosion in potential applications, the molecular interactions between GQDs and complex biomolecular systems, including potassium-ion (K+) channels, remain largely unknown. Here, we use molecular dynamics (MD) simulations and electrophysiology to study the interactions between GQDs and three representative K+ channels, which participate in a variety of physiological processes and are closely related to many disease states. Using MD simulations, we observed that GQDs adopt distinct contact poses with each of the three structurally distinct K+ channels. Our electrophysiological characterization of the effects of GQDs on channel currents revealed that GQDs interact with the extracellular voltage-sensing domain (VSD) of a Kv1.2 channel, augmenting current by left-shifting the voltage dependence of channel activation. In contrast, GQDs form a "lid" cluster over the extracellular mouth of inward rectifier Kir3.2, blocking the channel pore and decreasing the current in a concentration-dependent manner. Meanwhile, GQDs accumulate on the extracellular "cap domain" of K2P2 channels and have no apparent impact on the K+ flux through the channel. These results reveal a surprising multifaceted regulation of K+ channels by GQDs, which might help de novo design of nanomaterial-based channel probe openers/inhibitors that can be used to further discern channel function.

Identification of a unique endoplasmic retention motif in the Xenopus GIRK5 channel and its contribution to oocyte maturation.

G protein-activated inward-rectifying potassium (K+ ) channels (Kir3/GIRK) participate in cell excitability. The GIRK5 channel is present in Xenopus laevis oocytes. In an attempt to investigate the physiological role of GIRK5, we identified a noncanonical di-arginine endoplasmic reticulum (ER) retention motif (KRXY). This retention motif is located at the N-terminal region of GIRK5, coded by two small exons found only in X. laevis and X. tropicalis. These novel exons are expressed through use of an alternative transcription start site. Mutations in the sequence KRXY produced functional channels and induced progesterone-independent oocyte meiotic progression. The chimeric proteins enhanced green fluorescent protein (EGFP)-GIRK5-WT and the EGFP-GIRK5K13AR14A double mutant, were localized to the ER and the plasma membrane of the vegetal pole of the oocyte, respectively. Silencing of GIRK5 or blocking of this channel by external barium prevented progesterone-induced meiotic progression. The endogenous level of GIRK5 protein decreased through oocyte stages in prophase I augmenting by progesterone. In conclusion, we have identified a unique mechanism by which the expression pattern of a K+ channel evolved to control Xenopus oocyte maturation.

Ion channels as lipid sensors: from structures to mechanisms.

Ion channels play critical roles in cellular function by facilitating the flow of ions across the membrane in response to chemical or mechanical stimuli. Ion channels operate in a lipid bilayer, which can modulate or define their function. Recent technical advancements have led to the solution of numerous ion channel structures solubilized in detergent and/or reconstituted into lipid bilayers, thus providing unprecedented insight into the mechanisms underlying ion channel-lipid interactions. Here, we describe how ion channel structures have evolved to respond to both lipid modulators and lipid activators to control the electrical activities of cells, highlighting diverse mechanisms and common themes.

Cryo-EM analysis of PIP2 regulation in mammalian GIRK channels.

G-protein-gated inward rectifier potassium (GIRK) channels are regulated by G proteins and PIP2. Here, using cryo-EM single particle analysis we describe the equilibrium ensemble of structures of neuronal GIRK2 as a function of the C8-PIP2 concentration. We find that PIP2 shifts the equilibrium between two distinguishable structures of neuronal GIRK (GIRK2), extended and docked, towards the docked form. In the docked form the cytoplasmic domain, to which Gßγ binds, becomes accessible to the cytoplasmic membrane surface where Gßγ resides. Furthermore, PIP2 binding reshapes the Gßγ binding surface on the cytoplasmic domain, preparing it to receive Gßγ. We find that cardiac GIRK (GIRK1/4) can also exist in both extended and docked conformations. These findings lead us to conclude that PIP2 influences GIRK channels in a structurally similar manner to Kir2.2 channels. In Kir2.2 channels, the PIP2-induced conformational changes open the pore. In GIRK channels, they prepare the channel for activation by Gßγ.

A constricted opening in Kir channels does not impede potassium conduction.

The canonical mechanistic model explaining potassium channel gating is of a conformational change that alternately dilates and constricts a collar-like intracellular entrance to the pore. It is based on the premise that K+ ions maintain a complete hydration shell while passing between the transmembrane cavity and cytosol, which must be accommodated. To put the canonical model to the test, we locked the conformation of a Kir K+ channel to prevent widening of the narrow collar. Unexpectedly, conduction was unimpaired in the locked channels. In parallel, we employed all-atom molecular dynamics to simulate K+ ions moving along the conduction pathway between the lower cavity and cytosol. During simulations, the constriction did not significantly widen. Instead, transient loss of some water molecules facilitated K+ permeation through the collar. The low free energy barrier to partial dehydration in the absence of conformational change indicates Kir channels are not gated by the canonical mechanism.

Insight into the Selectivity of Kir3.2 toward Phosphatidylinositides.

Activation of G-protein-gated inwardly rectifying potassium channels (Kir3.x) requires the direct binding of phosphorylated phosphatidylinositides (PIPs). Previous studies have established that PIP isoforms activate Kir channels to varying degrees and the binding affinity between PIPs and Kir3.2 appears to be correlated with the level of activation. However, how individual residues contribute to the selectivity of Kir channels toward PIP isoforms is poorly understood. Here, we employ native mass spectrometry (MS) and fluorescent lipid binding assays to gain insight into the contribution of specific Kir3.2 residues binding to phospholipids. For the wild-type channel, we demonstrate the importance of membrane protein samples devoid of co-purified contaminants for protein-lipid binding studies and show that PIP(4,5)P2 cooperatively binds Kir3.2 with a Hill coefficient of 2.7. We also find lipid binding profiles determined from native MS and solution binding assays are in direct agreement. Point mutations of Kir3.2 residues that interact with PIPs distinctly alter selective lipid binding. The K64Q mutation results in altered binding profiles with the highest binding affinity for PIP(4,5)P2 with specific acyl chains. Mutation of R92 to Pro, a residue found in Kir6.2, results in promiscuous binding of PIP isoforms. Kir3.2 with the K194A mutation results in a distinct binding preference for PIP(3,4,5)P3 over other PIP isoforms. Taken together, our results underscore the utmost importance of protein quality for protein-lipid binding studies and show that a single mutation in Kir3.2 can alter the selectivity toward PIPs.

Codon Harmonization of a Kir3.1-KirBac1.3 Chimera for Structural Study Optimization.

The expression of functional, folded, and isotopically enriched membrane proteins is an enduring bottleneck for nuclear magnetic resonance (NMR) studies. Indeed, historically, protein yield optimization has been insufficient to allow NMR analysis of many complex Eukaryotic membrane proteins. However, recent work has found that manipulation of plasmid codons improves the odds of successful NMR-friendly protein production. In the last decade, numerous studies showed that matching codon usage patterns in recombinant gene sequences to those in the native sequence is positively correlated with increased protein yield. This phenomenon, dubbed codon harmonization, may be a powerful tool in optimizing recombinant expression of difficult-to-produce membrane proteins for structural studies. Here, we apply this technique to an inward rectifier K+ Channel (Kir) 3.1-KirBac1.3 chimera. Kir3.1 falls within the G protein-coupled inward rectifier K+ (GIRK) channel family, thus NMR studies may inform on the nuances of GIRK gating action in the presence and absence of its G Protein, lipid, and small molecule ligands. In our hands, harmonized plasmids increase protein yield nearly two-fold compared to the traditional 'fully codon optimized' construct. We then employ a fluorescence-based functional assay and solid-state NMR correlation spectroscopy to show the final protein product is folded and functional.

The small molecule GAT1508 activates brain-specific GIRK1/2 channel heteromers and facilitates conditioned fear extinction in rodents.

G-protein-gated inwardly-rectifying K+ (GIRK) channels are targets of Gi/o-protein-signaling systems that inhibit cell excitability. GIRK channels exist as homotetramers (GIRK2 and GIRK4) or heterotetramers with nonfunctional homomeric subunits (GIRK1 and GIRK3). Although they have been implicated in multiple conditions, the lack of selective GIRK drugs that discriminate among the different GIRK channel subtypes has hampered investigations into their precise physiological relevance and therapeutic potential. Here, we report on a highly-specific, potent, and efficacious activator of brain GIRK1/2 channels. Using a chemical screen and electrophysiological assays, we found that this activator, the bromothiophene-substituted small molecule GAT1508, is specific for brain-expressed GIRK1/2 channels rather than for cardiac GIRK1/4 channels. Computational models predicted a GAT1508-binding site validated by experimental mutagenesis experiments, providing insights into how urea-based compounds engage distant GIRK1 residues required for channel activation. Furthermore, we provide computational and experimental evidence that GAT1508 is an allosteric modulator of channel-phosphatidylinositol 4,5-bisphosphate interactions. Through brain-slice electrophysiology, we show that subthreshold GAT1508 concentrations directly stimulate GIRK currents in the basolateral amygdala (BLA) and potentiate baclofen-induced currents. Of note, GAT1508 effectively extinguished conditioned fear in rodents and lacked cardiac and behavioral side effects, suggesting its potential for use in pharmacotherapy for post-traumatic stress disorder. In summary, our findings indicate that the small molecule GAT1508 has high specificity for brain GIRK1/2 channel subunits, directly or allosterically activates GIRK1/2 channels in the BLA, and facilitates fear extinction in a rodent model.

Structural Determinants Mediating Tertiapin Block of Neuronal Kir3.2 Channels.

Tertiapin (TPN) is a 21 amino acid venom peptide from Apis mellifera that inhibits certain members of the inward rectifier potassium (Kir) channel family at a nanomolar affinity with limited specificity. Structure-based computational simulations predict that TPN behaves as a pore blocker; however, the molecular determinants mediating block of neuronal Kir3 channels have been inconclusive and unvalidated. Here, using molecular docking and molecular dynamics (MD) simulations with 'potential of mean force' (PMF) calculations, we investigated the energetically most favored interaction of TPN with several Kir3.x channel structures. The resulting binding model for Kir3.2-TPN complexes was then tested by targeted mutagenesis of the predicted contact sites, and their impact on the functional channel block was measured electrophysiologically. Together, our findings indicate that a high-affinity TPN block of Kir3.2 channels involves a pore-inserting lysine side chain requiring (1) hydrophobic interactions at a phenylalanine ring surrounding the channel pore and (2) electrostatic interactions with two adjacent Kir3.2 turret regions. Together, these interactions collectively stabilize high-affinity toxin binding to the Kir3.2 outer vestibule, which orients the ε-amino group of TPN-K21 to occupy the outermost K+ binding site of the selectivity filter. The structural determinants for the TPN block described here also revealed a favored subunit arrangement for assembled Kir3.x heteromeric channels, in addition to a multimodal binding capacity of TPN variants consistent with the functional dyad model for polybasic peptide pore blockers. These novel findings will aid efforts in re-engineering the TPN pharmacophore to develop peptide variants having unique and distinct Kir channel blocking properties.

Cholesterol Binding Sites in Inwardly Rectifying Potassium Channels.

Inwardly rectifying potassium (Kir) channels play a variety of critical cellular roles including modulating membrane excitability in neurons, cardiomyocytes and muscle cells, and setting the resting membrane potential, heart rate, vascular tone, insulin release, and salt flow across epithelia. These processes are regulated by a variegated list of modulators. In particular, in recent years, cholesterol has been shown to modulate a growing number of Kir channels. Subsequent to the discovery that members of the Kir2 subfamily were down-regulated by cholesterol, we have shown that members of several other Kir subfamilies were also modulated by cholesterol. However, not all cholesterol sensitive Kir channels were down-regulated by cholesterol. Our recent studies focused on three Kir channels: Kir2.1 (IRK1), Kir3.2^ (GIRK2^) and Kir3.4* (GIRK4*). Among these, Kir2.1 was down-regulated by cholesterol whereas Kir3.2^ and Kir3.4* were both up-regulated by cholesterol. Despite the opposite impact of cholesterol on these Kir3 channels compared to Kir2.1, putative cholesterol binding sites in all three channels were identified in equivalent transmembrane domains. Interestingly, however, there are intriguing differences in the specific residues that interact with the cholesterol molecule in these Kir channels. Here we compare and contrast the molecular characteristics of the putative cholesterol binding sites in the three channels, and discuss the potential implications of the differences for the impact of cholesterol on ion channels.

Structural basis for KCTD-mediated rapid desensitization of GABAB signalling.

The GABAB (γ-aminobutyric acid type B) receptor is one of the principal inhibitory neurotransmitter receptors in the brain, and it signals through heterotrimeric G proteins to activate a variety of effectors, including G-protein-coupled inwardly rectifying potassium channels (GIRKs)1,2. GABAB-receptor signalling is tightly regulated by auxiliary subunits called KCTDs, which control the kinetics of GIRK activation and desensitization3-5. However, the mechanistic basis for KCTD modulation of GABAB signalling remains incompletely understood. Here, using a combination of X-ray crystallography, electron microscopy, and functional and biochemical experiments, we reveal the molecular details of KCTD binding to both GABAB receptors and G-protein ßγ subunits. KCTDs associate with the receptor by forming an asymmetric pentameric ring around a region of the receptor carboxy-terminal tail, while a second KCTD domain, H1, engages in a symmetric interaction with five copies of Gßγ in which the G-protein subunits also interact directly with one another. We further show that KCTD binding to Gßγ is highly cooperative, defining a model in which KCTD proteins cooperatively strip G proteins from GIRK channels to induce rapid desensitization following receptor activation. These results provide a framework for understanding the molecular basis for the precise temporal control of GABAB signalling by KCTD proteins.

Solving a specificity mystery.

Differences in the kinetics of G protein activation can explain why only some receptors can activate potassium ion channels called GIRKs.